Myasthenia gravis is an autoimmune disease. In a patient suffering from the disease, autoantibodies are generated against epitopes on acetylcholine receptors at neuromuscular junctions. This autoimmune response impairs neuromuscular transmission. This impairment causes the muscular weakness and fatigability which characterize the disease.
In my patents, U.S. Pat. Nos. 4,202,875 and Re. 30,059, both of which are incorporated herein by reference, biochemical assays for the diagnosis of myasthenia gravis are described.
The assays in my above-cited patents involve immune precipitation, with anti-human immunoglobulin, of labeled acetylcholine receptor protein complexed with autoantibodies to the receptor protein from serum of a myasthenia gravis patient. The acetylcholine receptor protein (hereinafter "AChR") that is employed is part of a crude extract of mammalian muscle. The labeling of the AChR in these assays is accomplished by binding to the protein in the crude extract radioactively labeled, curaremimetic neurotoxins, such as Naja naja siamensis toxin and alpha-bungarotoxin. These toxins complex with the AChR by binding in the acetylcholine binding site on the protein.
While the assays described in my above-cited patents have found wdespread commercial application and are at present the preferred assays for diagnosing myasthenia gravis, they involve a number of drawbacks. The preferred source for the AChR utilized in these assays is muscle from amputated human legs. This tissue is in short supply and, on account of a variety of legal complications, difficult to obtain for commercial applications even when available. Further, it is possible to obtain from the tissue only very small quantities of AChR suitable for the assays because the protein is present at only very low concentration in even healthy, unamputated tissue and because substantial proteolytic degradation of the protein occurs unavoidably before a crude extract of the muscle containing the protein can be isolated from the amputated tissue. Other, more plentiful, mammalian-muscle sources for the AChR are available; but these other sources are not entirely free of the drawbacks, of sparse amount and proteolytic degradation, of the human leg-muscle source and, more importantly, the AChR from non-human sources is less acceptable for the assays because human autoantibodies to human AChR cross react poorly with AChR's from other mammals (e.g., to the extent of only 2.1% in the case of AChR from denervated rat muscle, a typical alternative to AChR from human leg muscle).
The limited supply of human muscle AChR suitable for immunochemical assay has made it impracticable to assay for myasthenia gravis-related autoantibody in human serum other than by the immune precipitation assay using crude extracts of muscle described in my above-cited patents. The ready availability of a more plentiful source of AChR, that reacts with human autoantibodies to human muscle AChR to substantially the same extent as human muscle AChR itself, would make practical other, more sensitive immunoassay techniques, such as solid-phase and enzyme-linked immunosorbant assay (ELISA) techniques, for the diagnosis of myasthenia gravis.
The severity of muscle weakness in myasthenia gravis patients does not correlate closely with the concentration of anti-AChR antibodies in their sera. This is probably largely due to the multiple, complex mechanisms by which these antibodies impair transmission (reduction of number of receptors by antigenic modulation, complement-mediated focal lysis, direct impairment of AChR function by bound antibody) as well as the nature of neuromuscular transmission (occurring in an all or none fashion with a large safety factor to insure transmission) and the complex adaptive mechanisms which are available to the body to compensate for impaired neuromuscular transmission (increasing acetylcholine release, increasing the area of neuromuscular junctions).
In myasthenia gravis patents, autoantibodies bind to various epitopes on various regions of AChR. One of these regions is called the "main immunogenic region". This region is located on the extracellular surface of alpha-subunits of the AChR. Another of these regions is the acetylcholine (hereinafter "ACh") binding sites, which are also located on the alpha-subunits. In the average myasthenia gravis patient, about half of the anti-AChR-autoantibodies are directed at the main immunogenic region. Autoantibodies which bind to the main immunogenic region can cause antigenic modulation and fix complement in vivo and thereby reduce the number of ACh receptors. Antibodies which are directed at the ACh binding sites would, at very low concentrations, be very effective at directly impairing AChR function in neuromuscular transmission. It would be useful if one could measure not only total anti-AChR antibody concentration in sera of myasthenia gravis patients but also the fractions of these antibodies which are directed at the main immunogenic region and the ACh-binding sites of AChR.
The assay of my above-cited patents depends on the labeling to high specific activity of the muscle AChR by complexing with it radioactively labeled toxins which bind in the ACh-binding site of the protein. It has not been possible to assay specifically for autoantibodies directed against the ACh-binding site with the assay of my above-cited patents because, in such assays, the binding site and nearby areas are blocked by toxin and, thereby, the epitopes in the binding site, and other epitopes near it, are inaccessible to binding by autoantibody. The invention described herein, based on the discovery of a source for large quantities of easily isolated AChR which, for practical purposes, is immunologically indistinguishable from human muscle AChR, makes feasible immunoassay methods which do not require blocking of some epitopes on the AChR from binding by autoantibodies. Thus, with the present invention, measurement is possible of not only the total concentration of anti-AChR autoantibody in the sera of a patient, permitting diagnosis, but also the fractions of these antibodies directed at pathologically significant sites on the AChR, permitting better characterization of the patient's disease and improved basis for planning therapy to treat the disease.
McAllister et al., Int. J. Cancer 20, 206-212 (1977) reported the establishment of a cell line, designated TE671, Subline No. 2, from a human cerebellar medulloblastoma. This and equivalent AChR-producing cell lines are referred to in this specification as "the TE671 Line".
Syapin et al., Brain Research 231, 365-377 (1982) have characterized the cells of the TE671 Line as possessing mammalian neuronal nicotinic acetylcholine receptors. It is thought in the art that the proteins of such receptors are immunologically substantially different from (i.e., have low immunological cross-reactivity with) the AChR against which myasthenia gravis autoantibodies react (i.e., that antibodies against one of neuronal AChR and muscle AChR have little or no cross-reactivity with the other) (Patrick and Stallcup, Proc. Natl. Acad. Sci. (U.S.A.) 74, 4689 (1977); Swanson et al., Proc. Natl. Acad. Sci. (U.S.A.) 80, 4532 (1983); Smith et al., J. Neuroscience 5, 2726 (1985)).
Hybridomas which secrete rat monoclonal antibodies, including that designated mAb 35, to the main immunogenic region of muscle AChR have been described by Tzartos et al., J. Biol. Chem. 256, 8635-8645 (1981) and Proc. Natl. Acad. Sci. U.S.A. 79, 188 (1982). Other hybridomas, including that designated mAb 64, which secrete monoclonal antibodies against muscle AChR have also been developed (Tzartos et al., J. Neuroimmunology, 10, 235-253 (1986)).